Electroporation device

ABSTRACT

An electroporation device produces electric signals that may be adjusted in response to a cover area of electrodes, so that the electric signals are tolerable when delivered to cells within the cover area. The electroporation device can include an applicator, a plurality of electrodes extending from the applicator, a power supply in electrical communication with the electrodes, and a guide member coupled to the electrodes. The electrodes are associated with a cover area. The power supply is configured to generate one or more electroporating signals to cells within the cover area. The guide member can be configured to adjust the cover area of the electrodes. In some embodiments, the electrical signals may include opposing waveforms that produce a resultant interference waveform to effectively target the cover area, and each waveform may be a unipolar waveform or a bipolar waveform.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/062,582, filed Oct. 24, 2013, which claims priority to U.S.Provisional Application No. 61/718,561, filed Oct. 25, 2012, U.S.Provisional Application No. 61/767,078, filed Feb. 20, 2013 and U.S.Provisional Application No. 61/791,968, filed Mar. 15, 2013, thecontents of all of which are fully incorporated herein.

TECHNICAL FIELD

This invention relates to an electroporation device configured todeliver one or more electroporating signals in a tolerable manner.

BACKGROUND

In the 1970's, it was discovered that electrical fields could be used tocreate pores in cells without causing permanent damage to the cell. Thisdiscovery made it possible for large molecules, ions, and water to beintroduced into a cell's cytoplasm through the cell wall. In someinstances, electroporation can be used in topical treatments, such asfor head and neck cancer, to introduce chemicals and other compoundsinto the tumor. During these procedures, the patient may not be undergeneral anesthesia so pain and involuntary muscle movement shouldpreferably be minimized.

Some electroporation devices can produce pulse trains that induceelectroporation within a cell's wall to allow the introduction of largemolecules, ions, and water into the cell's cytoplasm. However, theirelectric field or signal frequency (generally about 3.3 Hz) necessary tocreate the electroporation effect might cause the patient to experiencesignificant pain while receiving treatment. The pain may be at least inpart a result of an inverse effect that the frequency or electric fieldhas on skin impedance when an electromagnetic wave is traveling throughflesh. For example, skin impedance at 50 Hz is approximately32000.OMEGA. while skin impedance at 4000 Hz is reduced to approximately40.OMEGA. It has been observed that the higher the impedance, thegreater the pain.

Therefore, there is a need in the art for an electroporation device thatdelivers a strong enough pulse for delivering an agent for treatment,but prevents pain due to cell structure impedance.

SUMMARY

The present invention is directed to an electroporation devicecomprising an applicator, a plurality of electrodes extending from theapplicator, a power supply in electrical communication with theelectrodes, and a guide member coupled to the electrodes. The electrodesmay be associated with a cover area. The power supply may be configuredto generate one or more electroporating signals to cells within thecover area. The guide member may be configured to adjust the cover areaof the electrodes. The guide member may be slidably coupled to theapplicator.

The guide member may be slidably coupled to the applicator and theapplicator may be associated with an applicator end. Sliding the guidemember toward the applicator end may decrease the cover area.

The guide member may be slidably coupled to the applicator. Theapplicator may be associated with an applicator end. Sliding the guidemember away from the applicator end may increase the cover area.

At least a portion of the electrodes may be positioned within theapplicator in a conical arrangement. The one or more electroporatingsignals may be each associated with an electric field. The device mayfurther comprise a potentiometer coupled to the power supply andelectrodes. The potentiometer may be configured to maintain the electricfield substantially within a predetermined range.

The one or more electroporating signals may be each associated with anelectric field. The device may further comprise a potentiometer coupledto the power supply and the electrodes. The potentiometer may beconfigured to maintain the electric field to about 1300 V/cm.

The power supply may be associated with an output power. The device mayfurther comprise a potentiometer coupled to the power supply and theelectrodes. The potentiometer may be configured to adjust the outputpower in response to the cover area of the electrodes.

The power supply may be associated with an output power. The device mayfurther comprise a potentiometer coupled to the power supply and theelectrodes. The potentiometer may be configured to reduce the outputpower in response to a reduced cover area of the electrodes.

The power supply may be associated with an output power. The device mayfurther comprise a potentiometer coupled to the power supply and theelectrodes. The potentiometer may be configured to increase the outputpower in response to an increased cover area of the electrodes.

The one or more electroporating signals may be each associated with anelectric field. The device may further comprise a potentiometer coupledto the power supply and the electrodes. The potentiometer may beconfigured to maintain the electric field within a predetermined rangeso as to substantially prevent permanent damage in the cells within thecover area.

The one or more electroporating signals may be each associated with anelectrical field. The device may further comprise a potentiometercoupled to the power supply and the electrodes. The potentiometer may beconfigured to maintain the electrical field within a predetermined rangeso as to substantially minimize pain.

The power supply may provide a first electrical signal to a firstelectrode and a second electrical signal to a second electrode. Thefirst and second electrical signals may combine to produce a wave havinga beat frequency. The first and second electrical signals may each haveat least one of a unipolar waveform and a bipolar waveform. The firstelectrical signal may have a first frequency and a first amplitude. Thesecond electrical signal may have a second frequency and a secondamplitude. The first frequency may be different from or the same as thesecond frequency. The first amplitude may be different from or the sameas the second amplitude.

The power supply may be associated with an output power. The device mayfurther comprise a potentiometer coupled to the power supply and theelectrodes. The guide member may be slidably coupled to the applicator.The potentiometer may be configured to adjust the output power inresponse to the cover area of the electrodes.

The potentiometer may be configured to reduce the output power inresponse to a reduced cover area of the electrodes. The potentiometermay be configured to increase the output power in response to anincreased cover area of the electrodes. The one or more electroporatingsignals may be each associated with an electric field and thepotentiometer may be configured to maintain the electric field to about1300 V/cm.

The power supply may provide a first electrical signal to a firstelectrode and a second electrical signal to a second electrode. Thefirst and second electrical signals may combine to produce a wave havinga beat frequency. The first and second electrical signals may each haveat least one of a unipolar waveform and a bipolar waveform. The firstelectrical signal may have a first frequency and a first amplitude. Thesecond electrical signal may have a second frequency and a secondamplitude. The first frequency may be different from or the same as thesecond frequency. The first amplitude may be different from or the sameas the second amplitude.

The present invention is also directed to method of electroporatingcells using an electroporation device. The electroporation device maycomprise an applicator, a plurality of electrodes extending from theapplicator, a power supply in electrical communication with theelectrodes, and a guide member coupled to the electrodes. The electrodesmay be associated with a cover area. The power supply may be configuredto generate one or more electroporating signals to cells within thecover area. The guide member may be configured to adjust the cover areaof the electrodes.

The method may comprise administering selected molecules into the cellswithin the cover area, contacting the cells with the electrodes, anddelivering the one or more electroporating signals. The method mayfurther comprise adjusting the cover area of the electrodes.

The one or more electroporating signals may be each associated with anelectric field. Delivering the one or more electroporating signals mayfurther comprise maintaining the electrical field within a predeterminedrange so as to substantially prevent permanent damage in the cellswithin the cover area.

The one or more electroporating signals may be each associated with anelectrical field. Delivering the one or more electroporating signals mayfurther comprise maintaining the electrical field within a predeterminedrange so as to substantially minimize pain. The method may furthercomprise adjusting a temperature of the cells to about 4 .degree. C. toabout 45 .degree. C.

The present invention is also directed to an electroporation devicecomprising an applicator, a plurality of electrodes extending from theapplicator, a power supply in electrical communication with theelectrodes, and a camera coupled to the applicator and positionedadjacent the electrodes. The device may further comprise a device memoryin electronic communication with the camera. The device memory may beconfigured to store an electroporation treatment database.

The present invention is also directed to an electroporation devicecomprising an applicator, a plurality of electrodes extending from theapplicator, a power supply in electrical communication with theelectrodes, and a cooling/heating element coupled to the applicator andpositioned adjacent the electrodes. The power supply may provide a firstelectrical signal to a first electrode and a second electrical signal toa second electrode. The first and second electrical signals may combineto produce a wave having a beat frequency. The first and secondelectrical signals may each have at least one of a unipolar waveform anda bipolar waveform. The first electrical signal may have a firstfrequency and a first amplitude. The second electrical signal may have asecond frequency and a second amplitude. The first frequency may bedifferent from or the same as the second frequency. The first amplitudemay be different from or the same as the second amplitude.

The cooling/heating element may include a Peltier cooler. Thecooling/heating element may be configured to adjust a temperature oftumor cells to about 4 .degree. C. to about 45 .degree. C.

Other aspects of the invention will become apparent by consideration ofthe detailed description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross-sectional view of an applicator of the electroporationdevice according to one embodiment.

FIG. 2 is a perspective view of an electroporation device illustratingan applicator according to another embodiment.

FIG. 3 is a schematic of circuitry of the electroporation device of FIG.2.

FIG. 4 a is a perspective view of one embodiment of an applicator of theelectroporation device of FIG. 2, illustrating a disposable needle arraytip.

FIG. 4 b is a cross-sectional view of the applicator of FIG. 4 a.

FIG. 5 is a cross-sectional view of an applicator of the electroporationdevice of FIG. 2, illustrating a retractable shield.

FIG. 6 is a cut-away view of an applicator of the electroporation deviceaccording to yet another embodiment.

FIG. 7 is a plan view of an applicator of the electroporation deviceaccording to still another embodiment.

FIG. 8 is a schematic of one embodiment of an electrode needle array ofthe electroporation device of FIG. 2, illustrating partially insulatedelectrode needles.

FIG. 9 is a schematic of a 4.times.4 mapping array for needles of theelectroporation device of FIG. 2, illustrating 9 treatment zones.

FIG. 10 a is a schematic of a pulse sequence for a 2.times.2 treatmentzone of the electroporation device of FIG. 2.

FIGS. 10 b-10 d illustrate a pulse sequence for a 6-needle array of theelectroporation device of FIG. 2.

FIG. 11 is a graph plotting waveforms produced by the electroporationdevice of FIG. 2.

FIGS. 12 a-12 c are schematic illustrations of opposing unipolarwaveforms and the resultant unipolar interference waveform, produced bythe electroporation device of FIG. 2.

FIGS. 13 a-13 c are schematic illustrations of two waveforms and theresultant interference waveform, produced by the electroporation deviceof FIG. 2.

FIG. 14 shows photonic emission at 24 hours for cells mixed with DNAencoding luciferase.

FIG. 15 shows photonic emission at 24 hours for cells mixed with DNAencoding luciferase and then treated with interference electroporation.

FIG. 16 shows photonic emission of mice at day 1 following injectionwith plasmid DNA.

FIG. 17 shows photonic emission of mice at day 1 following injectionwith plasmid DNA and treatment with interference electroporation.

FIG. 18 shows photonic emission of mice at day 2 following injectionwith plasmid DNA.

FIG. 19 shows photonic emission of mice at day 2 following injectionwith plasmid DNA and treatment with interference electroporation.

FIG. 20 shows photonic emission of mice at day 5 following injectionwith plasmid DNA.

FIG. 21 shows photonic emission of mice at day 5 following injectionwith plasmid DNA and treatment with interference electroporation.

FIG. 22 shows photonic emission of mice at day 7 following injectionwith plasmid DNA.

FIG. 23 shows photonic emission of mice at day 7 following injectionwith plasmid DNA and treatment with interference electroporation.

FIG. 24 is a graph plotting time after plasmid DNA delivery and averagephoton emission.

FIG. 25 a shows representative hematoxylin and eosin (H&E) staining oftissue sections from mice injected with plasmid DNA; and FIG. 25 b showsrepresentative hematoxylin and eosin (H&E) staining of tissue sectionsfrom mice injected with plasmid DNA and receiving interferenceelectroporation treatment.

DETAILED DESCRIPTION

The inventors have discovered a new type of electroporation device thatcan cover or accommodate tumors of various sizes. The electroporationdevice may adjust electric signals in response to a cover area ofelectrodes, so that the pain from the electric signals may be tolerablewhen the electric signals are delivered to cells within the cover area.The electric signals may include opposing waveforms that produce aresultant interference waveform to effectively target the cover area.Each opposing waveform may be a unipolar waveform or a bipolar waveform.The resultant interference waveform may be shaped to the tumor and thevoltage may vary across the tumor, for example, less voltage at aperiphery of the tumor and more voltage at a central portion of thetumor.

Moreover, the electroporation device may include a camera to measure asize of the tumor and to better place the electrode needles.Furthermore, the electroporation device may include a cooling/heatingelement to adjust a temperature of a surface of the tumor. The inventionthus provides an apparatus and a method for the therapeutic applicationof electroporation while minimizing tissue damage and the painexperienced by the patient.

I) ELECTROPORATION DEVICE

The electroporation device 10 of the present invention includes ahousing 14 (FIG. 2) containing circuitry (FIG. 3), an electrodeapplicator 22, 200 removably coupled to the housing 14 (FIG. 2), and afoot pedal 26 coupled to the housing 14 and in electrical communicationwith the circuitry 18 (FIG. 3). A remote therapy activation connectionmay be provided to accommodate the foot pedal 26 for activating pulsesto the electrode applicator 22, 200. The foot pedal 26 may permit aphysician to activate the electroporation device 10 while freeing bothhands for positioning of the electrode applicator 22, 200 in a patient'stissue. Indicator lights for fault detection, power on, and completionof a therapy session may be provided for convenience. Other indicatorlights may be provided to positively indicate that the electrodeapplicator 22, 200 is connected to the electroporation device 10 and toindicate the type of needle array. A standby/reset button may beprovided to pause the electroporation device 10 and reset all functionsof the electroporation device 10 to a default state. A ready button maybe provided to prepare the electroporation device 10 for a therapysession. A “therapy in process” indicator light may indicate thatvoltage pulses are being applied to the electrode applicator 22, 200. Inaddition, the electroporation device 10 may have audio indicators forsuch functions as a button press, a fault state, commencement ortermination of a therapy session, indication of therapy in process, etc.In some embodiments, the electroporation device 10 can be coupled to afeedback sensor that detects heart beats. Applying pulses near the heartmay interfere with normal heart rhythms. By synchronizing application ofpulses to safe periods between beats, the possibility of suchinterference may be reduced.

Referring to FIG. 1, the illustrated electrode applicator 200 includes abody 30, a first electrode 34 having a first set of electrode needles38, and a second electrode 42 having a second set of electrode needles46. During operation, the user can manually manipulate the electrodeapplicator 22 to place the electrode needles 38, 46 in physical contactwith the target area of the tissue. The illustrated electroporationdevice 10 includes a potentiometer 228 that is configured to adjust anelectric output power in response to a cover area 220 of the electrodes34, 42, thereby maintaining an electric field of the electric signalssubstantially within a predetermined range.

A) Electrode Applicator

Referring to FIG. 1, in the illustrated embodiment, a guide member 204is coupled to the electrodes 34, 42. The electrodes 34, 42 areassociated with the cover area 220. A power supply is configured togenerate one or more electroporating signals 94, 110 to cells within thecover area 220. The guide member 204 is configured to adjust the coverarea 220 of the electrodes 34, 42. In the illustrated embodiment, theguide member 204 is in the form of a ring. In other embodiments, theguide member 204 can instead include portions of a ring, arcuatemembers, and the like that can suitably adjust the cover area 220 of theelectrodes 34, 42.

In the illustrated embodiment, the guide member 204 is coupled to theelectrode applicator 200. The electrode applicator 200 is associatedwith an applicator end 212, and sliding the guide member 204 toward theapplicator end 212 decreases the cover area 220 of the electrodes 34,42. For example, each electrode 34, 42 may be needle-shaped, and includespring tension at an end distal to the applicator end 212. In theillustrated embodiment, the electrodes 34, 42 are positioned within theelectrode applicator 200 in a conical arrangement. The conicalarrangement of the electrodes 200 is associated with an apex or tip 216,where individual electrodes 34, 42 are connected to one another in atight bundle, and the cover area 220 positioned away from the apex ortip 216. In some embodiments, the cover area 220 may assume anygeometric form, including, but not limited to, a circle associated witha diameter 224, an oval, an ellipse, a lens, a squircle, a polygon, asymbol, or a combination thereof.

In the illustrated embodiment, for example, as the guide member 204 ismoved toward the applicator end 212, the spring-tensioned electrodes 34,42 are drawn radially inward within the conical arrangement, therebyreducing the base diameter 224. On the other hand, sliding the guidemember 204 away from the applicator end 212 increases the base diameter224, thereby increasing the cover area 220. Although FIG. 1 illustratesthe guide member 204 as being slidably coupled to the electrodeapplicator 200, in other embodiments, the guide member 204 may becoupled to the electrode applicator 200 using other mechanisms.

In the illustrated embodiment, the potentiometer 228 is coupled to thepower supply and the electrodes 34, 42. The power supply is associatedwith an output power, and the potentiometer 228 is configured to adjustthe output power in response to the cover area 220 of the electrodes 34,42. For example, in the illustrated embodiment, sliding the guide member204 toward and away from the applicator end 212 can provide feedback tothe power supply regarding the associated base diameter 224 or coverarea 220, so that the output power can be adjusted accordingly. In theillustrated embodiment, the potentiometer 228 is electrically insulatedfrom the guide member 204 and supports the guide member 204 duringtravel. The potentiometer 228 is configured to reduce the output powerin response to a reduced base diameter 224 or cover area 220 of theelectrodes 34, 42, and increase the output power in response to anincreased base diameter 224 or cover area 220 of the electrodes 34, 42.In other embodiments, the potentiometer 228 may be configured to adjustthe output power using other mechanisms.

The electroporating signals 94, 110 are each associated with an electricfield, and in some embodiments the potentiometer 228 is configured tomaintain the electric field substantially within a predetermined rangeso as to substantially prevent permanent damage in the cells within thecover area 220 and to minimize the amount of pain experienced by theuser. For example, the potentiometer 228 can be configured to maintainthe electric field to about 1.300 kV/cm. In some embodiments, thepotentiometer is configured to maintain the electric field to at least375 V/cm, at least 450 V/cm, at least 525 V/cm, at least 600 V/cm, atleast 675 V/cm, at least 750 V/cm, at least 825 V/cm, at least 900 V/cm,at least 975 V/cm, at least 1.000 kV/cm, at least 1.075 kV/cm, at least1.150 kV/cm, or at least 1.225 kV/cm. In further embodiments, thepotentiometer 228 is configured to maintain the electric field to nomore than 1.300 kV/cm, no more than 1.225 kV/cm, no more than 1.150kV/cm, no more than 1.075 kV/cm, no more than 1.000 kV/cm, no more than925 V/cm, no more than 850 V/cm, no more than 775 V/cm, no more than 750V/cm, no more than 675 V/cm, no more than 600 V/cm, no more than 525V/cm, or no more than 450 V/cm. In other embodiments, the potentiometer228 may be configured to maintain the electric field as other values.

FIGS. 2 and 5 illustrate an electrode applicator of an electroporatingdevice according to another embodiment. This embodiment employs much ofthe same structure and has many of the same properties as the embodimentof the electroporation device described above in connection with FIG. 1.Accordingly, the following description focuses primarily upon thestructure and features that are different than the embodiment describedabove in connection with FIG. 1. Structure and features of theembodiment shown in FIG. 1 that correspond to structure and features ofthe embodiment of FIGS. 2 and 5 are designated hereinafter with likereference numbers.

The guide member 206 in this embodiment is a retractible shield. Theretractible shield 206 may be restricted by a friction O-ring (notshown) near a distal end of the body 30 of the electrode applicator 22,and can be slid fore and aft along the body 30 to protect or expose thefirst and second electrodes 34, 42. Thus, the retractible shield 206 isconfigured to adjust the cover area 208 of the first and secondelectrodes 34, 42 to accommodate tumors of various sizes.

Because it is possible for a number of different electrode applicator 22designs to be attached to the electroporation device 10, the electrodeapplicator 22 includes an electrically erasable programmable read-onlymemory (EEPROM) chip 50 (FIG. 3) with profile data stored thereon. Theprofile data is unique to each specific applicator and may includeinformation regarding the model, the make, the number of electrodespresent, and instructions for a desired treatment. In use, theelectroporation device 10 can read the EEPROM chip 50 to assure theproper settings are being used for each particular type of electrodeapplicator 22.

B) Integrated Wide-Angle Camera Applicator

Referring to FIG. 6, in the illustrated embodiment, a small or miniaturewide-angle camera 300 is embedded or integrated in the electrodeapplicator 22 between the electrode needles 38, 46. In some embodiments,this camera 300 may interface with an onboard electroporator software toacquire details regarding a tumor, such as type, size, shape, color,condition, and progression during electroporation therapy (EPT). Theelectroporator software may display an image transmitted from the camera300, so that a clinician or medical professional performing EPT candetermine tumor treatment coverage. In further embodiments, the tumormay be displayed with a visible grid. The clinician may then utilizevisual analysis algorithms to measure a size of the tumor and to betterplace the electrode needles 38, 46, thereby ensuring a completeelectroporation coverage. As explained below, in some embodiments, theelectroporator software may also record a patient number (notnecessarily including patient's personal information), dates oftreatments, waveform parameters, needle placement, and therapeutic agentdosage.

C) Integrated Thermoelectric Cooler/Heater Applicator

Referring to FIG. 7, in the illustrated embodiment, a small or miniaturethermoelectric cooling/heating element 400 is included or integrated inthe electrode applicator 22. The illustrated thermoelectriccooling/heating element 400 is located at a distal end of the electrodeapplicator 22. In some embodiments, the thermoelectric cooling/heatingelement 400 may provide non-contact or radiant pre-cooling/heating to asurface of the tumor. In other embodiments, however, the thermoelectriccooling/heating element 400 may provide contact cooling/heating to asurface of the tumor.

Lowering a temperature of the tumor cells before electroporation toabout 4 .degree. C. may improve the transfection of the therapeuticagent about ninefold. Thus, the thermoelectric cooling/heating element400 can reduce the temperature of the solid tumor body, and therebyimprove transfection. In some embodiments, the thermoelectriccooling/heating element 400 can reduce the temperature of the tumorcells before EPT to about 45 .degree. C., to about 44 .degree. C., toabout 43 .degree. C., to about 42 .degree. C., to about 41 .degree. C.,to about 40 .degree. C., to about 39 .degree. C., to about 38 .degree.C., to about 37 .degree. C., to about 36 .degree. C., about 35 .degree.C., about 34 .degree. C., about 33 .degree. C., about 32 .degree. C.,about 31 .degree. C., about 30 .degree. C., about 29 .degree. C., about28 .degree. C., about 27 .degree. C., about 26 .degree. C., about 25.degree. C., about 24 .degree. C., about 23 .degree. C., about 22.degree. C., about 21 .degree. C., about 20 .degree. C., about 19.degree. C., about 18 .degree. C., about 17 .degree. C., about 16.degree. C., about 15 .degree. C., about 14 .degree. C., about 13.degree. C., about 12 .degree. C., about 11 .degree. C., about 10.degree. C., about 9 .degree. C., about 8 .degree. C., about 7 .degree.C., about 6 .degree. C., about 5 .degree. C., or about 4 .degree. C. Insome embodiments, the thermoelectric cooling/heating element 400 canincrease the temperature of the tumor cells before electroporation toabout 38 .degree. C., about 39 .degree. C., about 40 .degree. C., about41 .degree. C., about 42 .degree. C., about 43 .degree. C., about 44.degree. C., or about 45 .degree. C. Thus, the thermoelectriccooling/heating element 400 can adjust the temperature of the tumorcells before EPT to about 4 .degree. C. to about 45 .degree. C., toabout 33 .degree. C. to about 45 .degree. C., to about 4 .degree. C. toabout 40 .degree. C., or to about 33 .degree. C. to about 40 .degree. C.

In some embodiments, the thermoelectric cooling/heating element 400 mayinclude a Peltier cooler. A Peltier cooler is a solid-state active heatpump creating a heat flux between a junction of two different types ofmaterials. The heat is thereby transferred from one material to theother, with consumption of electrical energy, depending on the directionof the current. In other embodiments, the thermoelectric cooling/heatingelement 400 may utilize other cooling mechanisms. In operation, theclinician may move the distal end of the electrode applicator 22 closeto the tumor before beginning EPT, and lower the temperature of thetumor tissue through radiant cooling. The reduced tumor temperature mayincrease the percentage of therapeutic agent transfer into the cells. Insome embodiments, the thermoelectric cooling/heating element 400 may bepowered by the same power supply as the electrode applicator 22.

D) Electroporation Treatment Database

In some Embodiments, an Electroporation Device Memory (not Shown) MayStore Data such as tumor type, photographic record of tumor size, color,shape, tumor progression, therapeutic agent dosage, electroporationparameters, and needle insertion placement. In further embodiments, thisdata may be collected into the electroporation device memory anddownloaded, e.g., periodically, via a wireless connection to a cloudstorage database (not shown). This may facilitate the creation of anovel database which can be developed to answer questions about futureEPT protocols or aspects of the treatment unknown at this time. In someembodiments, this information may not include patient personalinformation, but instead include a patient number. In furtherembodiments, patient sex, age, location, treating physician, tumor type,photographic record of tumor size, color, shape, tumor progression,therapeutic agent dosage, EPT parameters, needle insertion placement,etc., may be collected and included in the database.

E) Electrical Signals

The electroporation device 10 as disclosed herein is operable to providean unlimited variety of electric signals so long as the pain from theelectric signals is tolerable. In some embodiments, the electroporationdevice 10 is operable to separately apply pulses of high amplitudeelectric signals to at least two pairs of the first and secondelectrodes 34, 42. The electric signals may be applied proportionatelyto the distance between the electrodes of a pair to generate a nominalfield strength of about 10 V/cm to about 1500 V/cm in the cells andeffect introduction of selected molecules into the cells withoutpermanently damaging the cells. In some embodiments, the electricsignals may be applied simultaneously. In further embodiments, theelectric signals may be applied to some, but not all electrodes.

Referring to FIG. 11, in some embodiments, the electroporation device 10sends multiple, independent electric signals during operation toselected electrode needles 34, 42 that, when in contact with tissue, cancause electroporation in the cell wall. That is, a power supply (notshown) may provide a first electrical signal 94 to the first electrode34 and a second electrical signal 110 to the second electrode 42. Whenthe first and second electrodes 34, 42 are in electrical contact with abiological sample, the first electrical signal 94, which has a firstfrequency (corresponding to the wavelength 18 in FIG. 11), and thesecond electrical signal 110, which has a second frequency(corresponding to the wavelength 114 in FIG. 11), different or the samefrom the first frequency (the first and second signals may haveamplitudes that are different or the same), combine to produce aresultant wave 130 that may include a beat frequency and an embeddedfrequency to effect introduction of selected molecules into cells of thesample without permanently damaging the cells and minimizing pain. Inother embodiments, however, the power supply of the electroporationdevice 10 may adjust the electrical signals for all electrodes 34, 42 inunison. That is, the electrical signals for one electrode may not beindependent of other electrodes, and the electrical signals 94, 110 maynot produce a beat frequency.

The nature of the tissue, the size of the selected tissue, and itslocation determine the nature of the electric signals 94, 110 to begenerated. It is desirable that the field be as homogenous as possibleand of the correct amplitude. An excessive field strength may result inlysis of cells, whereas a low field strength may result in a reducedefficiency of delivering agents into the cell. This is especially truein the present invention where the resultant or resulting wave 130(e.g., the waveform experienced by the patient during therapy) is theresult of the interference of the first and second electrical signals94, 110. As such, any minor variances in the first and second signals94, 110 could result in major variances in the resulting wave 130.

As illustrated in FIG. 11, the first electrical signal 94 may include asinusoidal, cosinusoidal or pulsed electrical wave having a firstfrequency (corresponding to the wavelength 98 in FIG. 11) and the firstamplitude 102. The electrical signal may be monopolar or bi-polardependent upon the specific treatment being administered. In someembodiments, the first frequency is generally between about 500 Hz andabout 10,000 Hz. In other embodiments, the first frequency is betweenabout 600 Hz and about 9,000 Hz. In still other embodiments, the firstfrequency is between 700 Hz and about 8,000 Hz. In still otherembodiments, the first frequency is between about 800 Hz and about 7,000Hz. In still other embodiments, the first frequency is between about 900Hz and about 6,000 Hz. In still other embodiments, the first frequencyis between about 1,000 Hz and about 5,000 Hz. In still otherembodiments, the first frequency is between about 2,000 Hz and about4,000 Hz. Furthermore, the first amplitude 102 is generally betweenabout 150 V and about 3,000 V. In other embodiments, the first amplitude102 is between about 250 V and about 2,000 V. In still otherembodiments, the first amplitude 102 is between about 350 V and about1,000 V. In still other embodiments, the first amplitude 102 is betweenabout 450 V and about 900 V. In still other embodiments, the firstamplitude 102 is between about 550 V and about 800 V. In the case of apulsed electrical wave, the first electrical signal 94 may produce fromabout 500 pulses per second to about 10,000 pulses per second.

As illustrated in FIG. 11, the second electrical signal 110 may includea sinusoidal or cosinusoidal electrical wave having a second frequency(corresponding to the wavelength 114 in FIG. 11) different from thefirst frequency and a second amplitude 118 substantially similar to thefirst amplitude 102. The second electrical signal 110 may be monopolaror bi-polar dependent upon the type of treatment being administered. Insome embodiments, the second frequency is generally between about 500 Hzand about 10,000 Hz. In other embodiments, the second frequency isbetween about 600 Hz and about 9,000 Hz. In still other embodiments, thesecond frequency is between 700 Hz and about 8,000 Hz. In still otherembodiments, the second frequency is between about 800 Hz and about7,000 Hz. In still other embodiments, the second frequency is betweenabout 900 Hz and about 6,000 Hz. In still other embodiments, the secondfrequency is between about 1,000 Hz and about 5,000 Hz. In still otherembodiments, the second frequency is between about 2,000 Hz and about4,000 Hz. Furthermore, the second amplitude 118 is generally betweenabout 150 V and about 3,000 V. In other embodiments, the secondamplitude 118 is between about 250 V and about 2,000 V. In still otherembodiments, the second amplitude 118 is between about 350 V and about1,000 V. In still other embodiments, the second amplitude 118 is betweenabout 450 V and about 900 V. In still other embodiments, the secondamplitude 118 is between about 550 V and about 800 V. In the case of apulsed electrical wave, the second electrical signal 110 may producefrom about 500 pulses per second to about 10,000 pulses per second.

Illustrated in FIG. 11, the resultant wave 130 is the result of thecombined interference of the first electrical signal or wave 94 and thesecond electrical signal or wave 110. Governed by the laws of waveinterference, the resultant wave 130 may include a beat frequency,defined as the frequency of the oscillation of the envelope of theresultant wave 130 (corresponding to a wavelength 138 in FIG. 11). Theresultant wave 130 may also include an embedded frequency defined as thefrequency of the carrier wave (corresponding to a wavelength 142 in FIG.11). In the illustrated embodiment, the beat frequency is between about2 Hz and about 3,000 Hz. In other embodiments, the beat frequency isbetween about 50 Hz and about 2,000 Hz. In still other embodiments thebeat frequency is between about 100 Hz and about 1,000 Hz. In stillother embodiments, the beat frequency is between about 200 Hz and about900 Hz. In still other embodiments, the beat frequency is between about300 Hz and about 800 Hz. In still other embodiments, the beat frequencyis between about 400 Hz and about 700 Hz. Furthermore, the embeddedfrequency is from about 500 Hz to about 10,000 Hz. In other embodiments,the embedded frequency is between about 600 Hz and about 9,000 Hz. Instill other embodiments, the embedded frequency is between 700 Hz andabout 8,000 Hz. In still other embodiments, the embedded frequency isbetween about 800 Hz and about 7,000 Hz. In still other embodiments, theembedded frequency is between about 900 Hz and about 6,000 Hz. In stillother embodiments, the embedded frequency is between about 1,000 Hz andabout 5,000 Hz. In still other embodiments, the embedded frequency isbetween about 2,000 Hz and about 4,000 Hz. An amplitude 140 of theresultant wave 130 is generally equal to the sum of the amplitudes ofthe interfering waves (e.g., the first wave and the second wavecorresponding to the first and second electrical signals 94, 110). Inthe case of a pulsed wave, the beat frequency of the resultant wave 130is from about 2 pulses per second (Hz) to about 3,000 pulses per second(Hz).

It is to be understood that although the above described wave forms aresinusoidal or cosinusoidal in nature, square waves, saw tooth waves,step waves, and the like may also be produced by the electroporationdevice 10. Referring also to FIG. 12, each opposing waveform may be aunipolar waveform or a bipolar waveform. When each opposing waveform isunipolar (e.g., square wave) as illustrated in FIGS. 12 a and 12 b, theresult interference waveform is also unipolar as illustrated in FIG. 12c. The amplitude of the illustrated resultant wave is generally equal tothe sum of the amplitudes of the opposing waves, and the beat frequencymay be defined as the pulse frequency of the resultant square wave.

In some embodiments, the resultant wave 130 may be the result of theinterference of more than two wave forms. The specific waveformsproduced by the electroporation device 10 are designed to produce thedesired electroporation effect in the cell wall while minimizing theamount of pain experienced by the user, minimizing tissue damage, andproviding maximum pore formation for introducing an agent into a cell.

As illustrated in FIGS. 13 a-13 c, two separate waveforms (i.e., signals1 and 2) may combine or interfere such that the resultant waveform has aspecific shape. The shape of the resultant waveform may be similar to(or the same as) the shape of the tissue or cells targeted forelectroporation. Accordingly, the electroporation effect is directed tothe targeted tissue or cells and not surrounding or other tissues,thereby minimizing tissue damage in the user. The waveforms may bevaried such that the resultant waveform has any desired shape, andtherefore, the electroporation effect may be produced in tissues orareas of treatment having different shapes and sizes. Theelectroporation effect may be shaped to the tissue or area of treatment.

Additionally, the voltage at the intersection of the two waveforms maybe the sum of the voltages of the two waveforms. For example, if boththe first and second waveforms have a voltage of 650 V, then at theintersection of the first and second waveforms, the voltage would be1300 V. The waveforms may each have a lower voltage, but combine orinterfere to provide the higher voltage needed for maximum poreformation for introducing the agent into a cell. These lower voltagewaveforms have a higher frequency, which in turn, reduces impedancethrough the tissue, decreases tissue damage, and minimizes painexperienced by the user.

The waveforms may also combine such that the resultant waveform has ashape matching the targeted tissue and the voltage varies across thetargeted tissue. For example, if the targeted tissue is a tumor, theresultant waveform may have a shape similar to (or the same as) a shapeof the tumor, but because the voltage varies across the tumor shape,different portions of the tumor are exposed to more or less voltage(e.g., less voltage at a periphery portion of the tumor and highervoltage at a central portion of the tumor). The electroporation effectmay be shaped to the tissue or area of treatment while a spectrum ofvoltages is present in the tissue or area of treatment. This in turnallows the electroporation effect to be directional to minimize tissuedamage and pain experienced by the user while maximizing pore formationfor the introduction of the agent into the cell.

F) Signal Generation

Illustrated in FIG. 3, the circuitry 18 of the electroporation device 10includes an AC power module 54, a first waveform generator 58, a secondwaveform generator 62, and a control module 66. In some embodiments, thecircuitry 18 of the electroporation device 10 produces a beat wave 130(FIG. 11). The beat wave 130 is designed to produce electroporation inthe cell while minimizing the amount of pain experienced by the patient.

The AC power module 54 of the circuitry 18 receives electricity from apower source (e.g., from a wall socket, a generator, and the like),conditions the signal, and isolates the signal into multiple powersources to be used as electrical power throughout device. Morespecifically, the AC power module 54 produces a low-voltage DC powersupply 70 suitable for use by the control module 66, and a high voltagepower supply 74 (e.g., up to several thousand volts) suitable forwaveform generation. In the illustrated construction, the AC powermodule 54 utilizes a large toroidal transformer to isolate and conditionthe power source signal.

The AC power module 54 also includes a plurality of capacitors 78 tostore the high voltage power supply 74 for use by the first and secondwaveform generators 58, 62. In the illustrated embodiment, pulse widthmodulation is used to control the high voltage power supply 74 so itbetter accommodates therapy and delivery requirements.

The control module 66 of the interior circuitry 18 includes amicroprocessor 82 and a custom programmable logic array (PLA) 86. ThePLA 86 of the current invention independently controls the first andsecond waveform generators 58, 62. Stated differently, the PLA 86 ispre-programmed with multiple sets of waveform profiles, eachcorresponding to a unique therapy or treatment. During operation, themicroprocessor 82 sends a signal to the PLA 86 instructing it to producea particular waveform profile. The PLA 86 then independently controlsthe first and second waveform generators 58, 62 until the signal fromthe microprocessor 82 is stopped. The PLA 86 also receives feedback fromthe produced waveforms (e.g., through a voltage and current monitor 88),to assure the waveforms are within acceptable parameters. In the eventthe waveforms are unacceptable or a fault is detected, the PLA 86 mayshut down automatically.

Utilizing the PLA 86 independent of the microprocessor 82 ensures thatthe generated waveforms are not affected by the other services requiredby the microprocessor 82 during operation of the device. The separationalso ensures the system reacts immediately to any faults or error inputsin a safe fashion.

The microprocessor 82 acts as a system controller, sending and receivingsignals from various devices in the electroporation device 10. One suchfunction of the microprocessor 82 includes determining the type ofelectrode applicator 22 in use. When the electrode applicator 22 isconnected to the housing 14, the microprocessor 82 reads the profiledata stored in the EEPROM chip 50 and uses that data to determine theproper waveform profiles the PLA 86 should produce. The microprocessor82 also receives a signal from the foot pedal 26 and is capable ofoutputting data regarding the treatment to a printer or other outputdevice 90.

The first waveform generator 58 receives high voltage electrical powerfrom the capacitors 78 of the AC power module 54, along with input fromthe PLA 86, to produce a first electrical signal 94 having a firstfrequency (corresponding to a wavelength 98 in FIG. 11) and a firstamplitude 102 (see FIG. 11). In the illustrated construction, the firstwaveform generator 58 utilizes an insulated gate bipolar transistor orIGBT to produce the first electrical signal 94.

The first waveform generator 58 is electrically connected to asolid-state high-voltage relay 106 to control and output the firstelectrical signal 94 to the desired electrode needles 38 of the firstelectrode 34. In the illustrated embodiment, after the waveform isproduced (e.g., by the IGBT), the waveform may be checked by the PLA 86before being passed on by the high-voltage relay 106.

The second waveform generator 62 is substantially similar to the firstwaveform generator 58. The second waveform generator 62 receives highvoltage electrical power from the capacitors 78 of the AC power module54, along with input from the PLA 86, to produce a second electricalsignal 110 having a second frequency (corresponding to a wavelength 114in FIG. 11), different from the first frequency, and a second amplitude118. In the illustrated construction, the second waveform generator 62utilizes an insulated gate bipolar transistor or IGBT to produce thesecond electrical signal 110.

The second waveform generator 62 is electrically connected to asolid-state high-voltage relay 122 to control and output the secondelectrical signal 110 to the desired electrode needles 46 of the secondelectrode 42. In the illustrated embodiment, after the waveform isproduced (e.g., by the IGBT), the waveform may be checked by the PLA 86before being passed on by the high-voltage relay 122.

G) Needle Array

1) Disposable Needle Array Tips

The electroporation device 10 may include disposable needle array tips.The whole needle array shown in FIG. 5 may be disposable, including thecable and the connector. However, it may be more desirable to make theneedle array tip an independent component that is detachable from thebody 30 and the cable. Hence, a needle array tip may be disposed afteruse similar to the disposable needles used in injection of a fluid drug.Such disposable needle array tips can be used to eliminate possiblecontamination due to improper sterilization when reusing a needle arraytip.

FIG. 4 a shows one embodiment 1800 of the electroporation applicatoraccording to this aspect of the invention. The electroporationapplicator 1800 includes an applicator handle 1810, a detachable needlearray tip 1820, and an applicator cable 1812 connected to the applicatorhandle 1810. The detachable needle array tip 1820 can be engaged to anddetached from one end of the applicator handle 1810. When engaged to theapplicator handle 1810, the detachable needle array tip 1820 can receiveelectrical signals from the electroporation device 10 through theapplicator cable 1812.

FIG. 4 b shows structure details of the applicator handle 1810 and thedetachable needle array tip 1820. The applicator handle 1810 includes amain body 1811A and a distal end 1811B formed on one end of the mainbody 1811A. The other end of the main body 1811A is connected to theapplicator cable (not shown, see FIG. 4 a). The main body 1811A includestwo or more conducting wires 1815 for transmitting electrical signals tothe detachable needle array tip 1820. These signals may include needlevoltage setpoint, pulse length, pulse shape, the number of pulses, andswitching sequence. In one embodiment, when the detachable needle arraytip 1820 is used to deliver a liquid substance, one or more electrodeneedles may be made hollow for transmitting the liquid substance and oneor more liquid channels may be accordingly implemented in the applicatorhandle 1810. The liquid channel may be integrated with one of theconducting wires 1815 by, for example, using a metal-coated plastic tubeor a metal tube. Alternatively, the liquid substance may be delivered toa target by using a separate device, for example, prior to applicationof the electrical pulses. The distal end 1811B has an opening 1813 forengaging the needle array tip 1820. A plurality of connector holes 1814are formed for receiving connector pins in the detachable needle arraytip 1820.

The detachable needle array tip 1820 has a plurality of electrodeneedles 1822 forming a desired needle array, a support part 1823A thatholds the electrode needles 1822, and a connector part 1823B forengaging to the applicator handle 1810. In one embodiment, when thedetachable needle array tip 1820 is also used to deliver the liquidsubstance, at least one electrode needle is hollow and is connected to aliquid channel in the applicator handle 1810 for receiving the liquidsubstance. The connector part 1823B is shaped to be inserted into theopening 1813 in the distal end 1811B of the applicator handle 1810. Alocking or snapping mechanism may be optionally implemented to securethe detachable needle array tip 1820 to the applicator handle 1810. Aplurality of connector pins 1825 corresponding to the electrode needles1822 are formed in the connector part 1823B for engaging to therespective connector holes 1814 in the distal end 1811B.

The detachable needle array tip 1820 may include a contamination shield1824 formed on the support part 1823A for preventing the applicatorhandle 1810 from directly contacting any substance during anelectroporation process. A removable plastic cover 1826 may also beformed on the support part 1823A to seal the electrode needles 1822 andmaintain the sterility of the needles 1822 prior to use.

In some embodiments, the applicator handle 1810 may be configured toreceive a detachable needle array tip 1820 with other numbers ofelectrode needles 1822. In further embodiments, an electricalidentification element or EEPROM 50 may be implemented to allow theelectroporation device 10 of FIG. 2 to determine the number of theelectrode needles in an attached needle array tip. This identificationelement 50 may also be configured to generate proper electrical signalparameters corresponding to an identified needle array tip. A desiredneedle array addressing scheme may be selected accordingly to addressthe electrode needles.

2) Needle Arrays with Partially Insulated Electrode Needles

The electroporation device 10 may include needle arrays with partiallyinsulated electrode needles. Each electrode needle in the fixed anddisposable needle arrays shown in FIGS. 1, 4 a, 4 b, 5, 6, 7, and 8 maybe partially covered with an insulator layer in such a way that only adesired amount of the tip portion is exposed. The pulsed electric fieldsgenerated by such a partially insulated needle array are primarilyconcentrated in regions between and near the exposed tip portions of theelectrode needles during a treatment, and are small in regions betweenand near the insulated portions. A partially insulated needle array canbe used to confine the electroporation in a targeted area with a tumorand significantly shield the skin and tissues beyond the target areafrom the electroporation process. This provides protection to theuninvolved skin and tissues, which are at risk because certain drugs maycause undesired or even adverse effects when injected into uninvolvedsurface tissue above the target area.

FIG. 8 shows one embodiment of a partially insulated needle array 1900.A support portion 1910 is provided to hold multiple electrode needles1920 in a predetermined array pattern. Each electrode needle 1920 has abase portion 1922 that is covered with a layer of electricallyinsulating material such as Teflon and a tip portion 1924 that isexposed. When electrical voltages are applied to the electrode needles1920, the generated electrical fields in regions among and near theexposed tip portions 1924 are sufficiently strong to causeelectroporation but the electrical fields in regions among and near theinsulated base portions 1922 are either negligibly small or completelydiminished so that electroporation cannot be effected due to theshielding of the insulation. Therefore, electroporation is localized orconfined in regions where the exposed tip portions 1924 are positioned.

The lengths of the insulated base portion 1922 and the exposed tipportion 1924 may be predetermined or may be adjustable based on thelocation of a specific target area in a body part. In oneimplementation, each needle electrode may be pre-wrapped with a suitableinsulating layer to cover most of the electrode needle with a minimalusable exposed tip portion. A user may adjust a desired amount of theexposed tip portion as needed in a treatment.

The partially insulated electrode needles shown in FIG. 8 can be usedfor both the fixed needle array as shown in FIG. 5 and the disposableneedle array shown in FIGS. 4 a and 4 b.

3) Needle Array Addressing

The electroporation device 10 of FIG. 2 is designed to accommodateelectrode applicators 22 having varying numbers of electrode needles 38,46. Accordingly, an addressing scheme has been developed that, in thepreferred embodiment, permits addressing up to 16 different needles,designated A through P, forming up to 9 square treatment zones andseveral types of enlarged treatment zones. A treatment zone comprises atleast 4 needles in a configuration of opposing pairs that are addressedduring a particular pulse. During a particular pulse, two of the needlesof a treatment zone are of positive polarity and two are of negativepolarity.

FIG. 9 shows a preferred 4.times.4 mapping array for needles forming 9square treatment zones numbered from the center and proceeding outwardradially and then clockwise. In the preferred embodiment, this mappingarray defines 4-needle, 6-needle, 8-needle, 9-needle, and 16-needleelectrode configurations. A 4-needle electrode comprises needles placedin positions F, G, K, and J (treatment zone 1). A 9-needle electrodecomprises needles placed in positions defining treatment zones 1-4. A16-needle electrode comprises needles placed in positions definingtreatment zones 1-9.

FIG. 10 a shows a pulse switching sequence for a 2.times.2 treatmentzone or mapping array in accordance with one embodiment of theinvention. During any of four pulses comprising a cycle, opposing pairsof needles are respectively positively and negatively charged, as shown.Other patterns of such pairs are possible, such as clockwise orcounterclockwise progression. For example, for a 9-needle electrodeconfiguration, a preferred cycle comprises 16 pulses (4 treatment zonesat 4 pulses each). For example, for a 16-needle electrode configuration,a preferred cycle comprises 36 pulses (9 treatment zones at 4 pulseseach).

A 6-needle electrode can comprise a circular or hexagonal array as shownin FIGS. 10 b-10 d. Alternatively, a 6-needle electrode can be definedas a subset of a larger array, such as is shown in FIG. 9. For example,with reference to FIG. 9, a 6-needle electrode can be defined as a2.times.3 rectangular array of needles placed in positions definingtreatment zones 1-2 (or any other linear pair of treatment zones), or ahexagonal arrangement of needles B, G, K, N, I, E (or any other set ofpositions defining a hexagon) defining an enlarged treatment zone (shownin dotted outline in FIG. 9). Similarly, an 8-needle electrode cancomprise an octagon, or a subset of the larger array shown in FIG. 9.For example, with reference to FIG. 9, an 8-needle electrode can bedefined as a 2.times.4 array of needles placed in positions definingtreatment zones 1, 2 and 6 (or any other linear triplet of treatmentzones), or an octagonal arrangement of needles B, C, H, L, O, N, I, E(or any other set of positions defining an octagon) defining an enlargedtreatment zone.

FIGS. 10 b-10 d show a hexagonal arrangement and one possible activationsequence. FIG. 10 b shows a first sequence, in which needles G and K arepositive and needles I and E are negative during a first pulse, and havereversed polarities during a next pulse; needles B and N, shown indotted outline, are inactive. FIG. 10 c shows a second sequence, inwhich needles K and N are positive and needles E and B are negativeduring a first pulse, and have reversed polarities during a next pulse;needles G and I are inactive. FIG. 10 d shows a third sequence, in whichneedles N and I are positive and needles B and G are negative during afirst pulse, and have reversed polarities during a next pulse; needles Kand E are inactive. In some embodiments, a total of 6 pulses may beapplied in a cycle of sequences. A similar activation sequence can beused for an octagonal arrangement, which may apply other numbers ofpulses.

Regardless of physical configuration, the preferred embodiments of theinvention may use at least two switched pairs of electrodes (forexample, as shown in FIG. 10 a) in order to achieve a relatively uniformelectric field in tissue undergoing EPT. The electric field intensityshould be of sufficient intensity to effect the process ofelectroporation, to allow incorporation of a treatment agent.

H) “Sweet Spot” Manipulation

The electroporation device 10 may produce a “sweet spot” during use. The“sweet spot” can be defined as the area in the tissue where the firstelectric signal 94 interferes with the second electric signal 110 toproduce the resultant wave 130. In some embodiments, the electroporationdevice 10 is designed to move the “sweet spot” with respect to theneedles 38, 46 of the electrode applicator 22. More specifically, theelectroporation device 10 may adjust the amplitude, frequency, and pulsetime of the signals being produced at each individual electrode needle38, 46 to move, with respect to the electrode needles 38, 46, the exactlocation where the resultant wave 130 is produced in the tissue. Assuch, the device 10 is able to treat multiple areas of the tissuewithout the need to continuously move the applicator 22. Although aunipolar resultant result interference waveform can effectively targetthe sweet spot, a bipolar resultant interference waveform may moreeffectively target or move the sweet spot compared to a unipolarresultant interference waveform, depending on the usage requirements orpreferences for the particular electroporation device 10.

II) THERAPEUTIC METHOD

The electroporation device 10 may be used in a therapeutic method. Thetherapeutic method of the invention includes electrotherapy, alsoreferred to herein as electroporation therapy (EPT), for the delivery ofan agent or molecule to a cell or tissue. The term “agent” or “molecule”as used herein refers to, for example, drugs (e.g., chemotherapeuticagents), nucleic acids (e.g., polynucleotides), peptides, andpolypeptides, including antibodies. The term polynucleotides includeDNA, cDNA, and RNA sequences. It should be understood that theelectroporation of tissue can be performed in vitro, in vivo, or exvivo. Electroporation can also be performed utilizing single cells,e.g., single cell suspensions, in vitro, or ex vivo in cell culture.

Drugs contemplated for use in the method of the invention are typicallychemotherapeutic agents having an antitumor or cytotoxic effect. Suchdrugs or agents include bleomycin, neocarcinostatin, suramin,doxorubicin, carboplatin, taxol, mitomycin C, and cisplatin. Otherchemotherapeutic agents will be known to those of ordinary skill in theart (see, for example, The Merck Index). In addition, “membrane-acting”agents are also included in the method of the invention. These agentsmay also be agents as listed above, or alternatively, agents which actprimarily by damaging the cell membrane. Examples of membrane-actingagents include N-alkylmelamide and para-chloro mercury benzoate. Thechemical composition of the agent will dictate the most appropriate timeto administer the agent in relation to the administration of theelectric pulse. For example, while not wanting to be bound by aparticular theory, it is believed that a drug having a low isoelectricpoint (e.g., neocarcinostatin, IEP=3.78), would likely be more effectiveif administered post-electroporation in order to avoid electrostaticinteraction of the highly charged drug within the field. Further, suchdrugs as bleomycin, which have a very negative log P, (P being thepartition coefficient between octanol and water), are very large in size(MW=1400), and are hydrophilic, thereby associating closely with thelipid membrane, diffuse very slowly into a tumor cell and are typicallyadministered prior to or substantially simultaneous with the electricpulse. In addition, certain agents may require modification in order toenter the cell allow more efficiently. For example, an agent such astaxol can be modified to increase solubility in water which would allowit to enter the cell more efficiently. Electroporation facilitates entryof bleomycin or other similar drugs into the tumor cell by creatingpores in the cell membrane.

In one embodiment, the invention provides a method of electroporatingcells using the electroporation device 10, comprising administeringselected molecules into the cells within the cover area 220, contactingthe cells with the electrodes 34, 42, and the electroporating signals94, 110. In some embodiments, the cover area 220 of the electrodes 34,42 may be adjusted before contacting the cells with the electrodes 34,42. In further embodiments, the electric field associated with theelectroporating signals 94, 110 may be maintained within a predeterminedrange so as to substantially prevent permanent damage in the cellswithin the cover area 220, and to minimize pain.

In another embodiment, the invention provides a method for thetherapeutic application of electroporation to a tissue of a subject forintroducing molecules into cells therein, comprising providing an arrayof electrodes, at least one of the electrodes having a needleconfiguration for penetrating tissue; inserting the needle electrodeinto selected tissue for introducing molecules into the tissue;positioning a second electrode of the array of electrodes in conductiverelation to the selected tissue; applying a first electric signal to thefirst electrode and applying a second electric signal to the secondelectrode such that a resultant electrical signal or wave is formed inthe tissue from the wave interference between the first wave and thesecond wave. The resultant wave has a beat frequency sufficient to causeelectroporation in the cell wall, and the embedded frequency issufficient to minimize pain.

In addition to minimizing pain and tissue damage, the method of theinvention may increase the uptake of the agent by cells relative to amethod that does not employ EPT. Uptake or introduction of the agent tothe cells may be increased by about 0.5-fold to about 50-fold, about0.5-fold to about 45-fold, about 0.5-fold to about 40-fold, about0.5-fold to about 35-fold, about 0.5-fold to about 30-fold, about0.5-fold to about 25-fold, about 1-fold to about 50-fold, about 1.5-foldto about 50-fold, about 2-fold to about 50-fold, about 2.5-fold to about50-fold, or about 3-fold to about 50-fold. Uptake of the agent by thecells may be increased by 0.5-fold to about 6-fold, about 0.75-fold toabout 6-fold, about 1-fold to about 6-fold, about 1.25-fold to about6-fold, about 1.5-fold to about 6-fold, about 1.75-fold to about 6-fold,about 2-fold to about 6-fold, about 2.25-fold to about 6-fold, about2.5-fold to about 6-fold, about 2.75-fold to about 6-fold, about 3-foldto about 6-fold, about 3.25-fold to about 6-fold, about 0.5-fold toabout 5.75-fold, about 0.5-fold to about 5.5-fold, about 0.5-fold toabout 5.25-fold, about 0.5-fold to about 5-fold, about 0.5-fold to about4.75-fold, about 0.5-fold to about 4.5-fold, about 0.5-fold to about4.25-fold, about 0.5-fold to about 4-fold, or about 0.5-fold to about3.75-fold. Uptake of the agent by the cells may also be increased byabout 0.75-fold to about 5.75-fold, about 1-fold to about 5.5-fold,about 1.25-fold to about 5.25-fold, about 1.5-fold to about 5-fold,about 1.75-fold to about 4.75-fold, about 2-fold to about 4.5-fold,about 2.25-fold to about 4.25-fold, about 2.5-fold to about 4-fold orabout 2.75-fold to about 3.75-fold.

Uptake of the agent by the cells may be increased by about 18-fold toabout 30-fold, about 18-fold to about 29-fold, about 18-fold to about28-fold, about 18-fold to about 27-fold, about 18-fold to about 26-fold,about 18-fold to about 24-fold, about 19-fold to about 30-fold, about20-fold to about 30-fold, about 21-fold to about 30-fold, about 22-foldto about 30-fold or about 23-fold to about 30-fold. Uptake of the agentby the cells may also be increased by about 19-fold to about 29-fold,about 20-fold to about 28-fold, about 21-fold to about 27-fold, about22-fold to about 26-fold, about 23-fold to about 25-fold, or about23-fold to about 24-fold. Uptake of the agent by the cells may beincreased by about 18-fold, about 19-fold, about 20-fold, about 21-fold,about 22-fold, about 23-fold, about 24-fold, about 25-fold, about26-fold, about 27-fold, about 28-fold, about 29-fold, or about 30-fold.

In another embodiment, uptake or introduction of the agent to the cellsmay be increased by about 50% to about 5000%, about 50% to about 4500%,about 50% to about 4000%, about 50% to about 3500%, about 50% to about3000%, about 50% to about 2500%, about 100% to about 5000%, about 150%to about 5000%, about 200% to about 5000%, about 250% to about 5000%, orabout 300% to about 5000%. Uptake of the agent by the cells may beincreased by about 50% to about 600%, about 75% to about 600%, about100% to about 600%, about 125% to about 600%, about 150% to about 600%,about 175% to about 600%, about 200% to about 600%, about 225% to about600%, about 250% to about 600%, about 275% to about 600%, about 300% toabout 600%, about 325% to about 600%, about 50% to about 575%, about 50%to about 550%, about 50% to about 525%, about 50% to about 500%, about50% to about 475%, about 50% to about 450%, about 50% to about 425%,about 50% to about 400%, or about 50% to about 375%. Uptake of the agentby the cells may also be increased by about 75% to about 575%, about100% to about 550%, about 125% to about 525%, about 150% to about 500%,about 175% to about 475%, about 200% to about 450%, about 225% to about425%, about 250% to about 400%, or about 275% to about 375%. Uptake ofthe agent by the cells may be increased by about 345%, about 346%, about347%, about 348%, about 349%, about 350%, about 351%, about 352%, about353%, about 354%, about 355%, about 356%, about 357%, about 358%, about359%, about 360%, about 361%, about 363%, about 364%, about 365%, about366%, about 367%, about 368%, about 369%, about 370%, about 371%, about372%, about 373%, about 374%, about 375%, about 376%, about 377%, about378%, about 379%, or about 380%.

Uptake of the agent by the cells may also be increased by about 1800% toabout 3000%, about 1800% to about 2900%, about 1800% to about 2800%,about 1800% to about 2700%, about 1800% to about 2600%, about 1800% toabout 2500%, about 1800% to about 2400%, about 1900% to about 3000%,about 2000% to about 3000%, about 2100% to about 3000%, about 2200% toabout 3000%, or about 2300% to about 3000%. Uptake of the agent by thecells may be increased by about 1850% to about 2950%, about 1900% toabout 2900%, about 1950% to about 2850%, about 2000% to about 2800%,about 2050% to about 2750%, about 2100% to about 2700%, about 2150% toabout 2650%, about 2200% to about 2600%, about 2250% to about 2550%,about 2300% to about 2500%, or about 2350% to about 2450%. Uptake of theagent by the cells may be increased by about 2300%, about 2310%, about2320%, about 2330%, about 2340%, about 2350%, about 2360%, about 2361%,about 2362%, about 2363%, about 2364%, about 2365%, about 2366%, about2367%, about 2368%, about 2369%, about 2370%, about 2371%, about 2372%,about 2373%, about 2374%, about 2375%, about 2376%, about 2377%, about2378%, about 2379%, about 2380%, about 2381%, about 2382%, about 2383%,about 2384%, about 2385%, about 2386%, about 2387%, about 2388%, about2389% or about 2390%.

Accordingly, the method of the invention minimizes the pain experiencedby the subject by decreasing impedance due to cell structure (e.g.,skin) while increasing the efficiency of introducing the agent into acell(s). The method of the invention advantageously provides effectivedelivery of the agent to within the cells, but unlike otherelectrotherapy methods, minimizes (or reduces) the pain experienced bythe subject due to impedance.

It may be desirable to modulate the expression of a gene in a cell bythe introduction of a molecule by the method of the invention. The term“modulate” envisions for example the suppression of expression of a genewhen it is over-expressed, or augmentation of expression when it isunder-expressed. Where a cell proliferative disorder is associated withthe expression of a gene, nucleic acid sequences that interfere with thegene's expression at the translational level can be used. This approachutilizes, for example, antisense nucleic acid, ribozymes, or triplexagents to block transcription or translation of a specific mRNA, eitherby masking that mRNA with an antisense nucleic acid or triplex agent, orby cleaving it with a ribozyme.

It is to be understood that the above described treatment may beutilized on various forms of solid cancer types, such as, but notlimited to, sarcomas, carcinomas, and lymphomas. Solid tumors may belocated throughout the body such as in the neck, lungs, skin, brain,prostate, liver, pancreatic, gall bladder, stomach, and lymph nodes.These tumors may further metastasize to other locations throughout thebody. The electroporation device 10 can be used to introduce agents suchas bleomycin to kill the tumor by necrosis and further stimulate theimmune system to prevent metastasis.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (Weintraub, ScientificAmerican, 262:40, 1990). In the cell, the antisense nucleic acidshybridize to the corresponding mRNA, forming a double-stranded molecule.The antisense nucleic acids interfere with the translation of the mRNA,since the cell will not translate a mRNA that is double-stranded.Antisense oligomers of about 15 nucleotides are preferred, since theyare easily synthesized and are less likely to cause problems than largermolecules when introduced into the target cell. The use of antisensemethods to inhibit the in vitro translation of genes is well known inthe art (Marcus-Sakura, Anal. Biochem., 172:289, 1988).

Use of an oligonucleotide to stall transcription is known as the triplexstrategy since the oligomer winds around double-helical DNA, forming athree-strand helix. Therefore, these triplex compounds can be designedto recognize a unique site on a chosen gene (Maher, et al., AntisenseRes. and Dev., 1(3):227, 1991; Helene, C., Anticancer Drug Design,6(6):569, 1991).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

There are two basic types of ribozymes, namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that the sequence will occur exclusively in the target mRNAspecies. Consequently, 18-based recognition sequences are preferable toshorter recognition sequences. Therefore, “hammerhead”-type ribozymesare preferable to tetrahymena-type ribozymes for inactivating a specificmRNA species.

The invention also provides gene therapy for the treatment of cellproliferative or immunologic disorders mediated by a particular gene orabsence thereof. Such therapy would achieve its therapeutic effect byintroduction of a specific sense or antisense polynucleotide into cellshaving the disorder. Delivery of polynucleotides can be achieved using arecombinant expression vector such as a chimeric virus, or thepolynucleotide can be delivered as “naked” DNA for example.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). When the subject is a human, a vector such as thegibbon ape leukemia virus (GaLV) can be utilized. A number of additionalretroviral vectors can incorporate multiple genes. All of these vectorscan transfer or incorporate a gene for a selectable marker so thattransduced cells can be identified and generated.

Therapeutic peptides or polypeptides may also be included in thetherapeutic method of the invention. For example, immunomodulatoryagents and other biological response modifiers can be administered forincorporation by a cell. The term “biological response modifiers” ismeant to encompass substances which are involved in modifying the immuneresponse. Examples of immune response modifiers include such compoundsas lymphokines Lymphokines include tumor necrosis factor, interleukins1, 2, and 3, lymphotoxin, macrophage activating factor, migrationinhibition factor, colony stimulating factor, and alpha-interferon,beta-interferon, and gamma-interferon and their subtypes.

Also included are polynucleotides which encode metabolic enzymes andproteins, including antiangiogenesis compounds, e.g., Factor VIII orFactor IX. The macromolecule of the invention also includes antibodymolecules. The term “antibody” as used herein is meant to include intactmolecules as well as fragments thereof, such as Fab and F(ab′).sub.2.

Administration of a drug, polynucleotide or polypeptide, in the methodof the invention can be, for example, parenterally by injection, rapidinfusion, nasopharyngeal absorption, dermal absorption, and orally. Inthe case of a tumor, for example, a chemotherapeutic or other agent canbe administered locally, systemically, or directly injected into thetumor. When a drug, for example, is administered directly into thetumor, it is advantageous to inject the drug in a “fanning” manner. Theterm “fanning” refers to administering the drug by changing thedirection of the needle as the drug is being injected or by multipleinjections in multiple directions like opening up of a hand fan, ratherthan as a bolus, in order to provide a greater distribution of drugthroughout the tumor. As compared with a volume that is typically usedin the art, it is desirable to increase the volume of thedrug-containing solution, when the drug is administered (e.g., injected)intratumorally, in order to ensure adequate distribution of the drugthroughout the tumor. For example, in the EXAMPLES using mice herein,one of skill in the art typically injects 50 .mu.l of drug-containingsolution, however, the results are greatly improved by increasing thevolume to 150 .mu.l. In human clinical studies, approximately 20 mlwould be injected to ensure adequate perfusion of the tumor. Preferably,the injection should be done very slowly all around the base and byfanning Although the interstitial pressure is very high at the center ofthe tumor, it is also a region where very often the tumor is necrotic.

Preferably, the molecule is administered substantially contemporaneouslywith the electroporation treatment. The term “substantiallycontemporaneously” means that the molecule and the electroporationtreatment are administered reasonably close together with respect totime, i.e., before the effect of the electrical pulses on the cellsdiminishes. The administration of the molecule or therapeutic agentdepends upon such factors as, for example, the nature of the tumor, thecondition of the patient, the size and chemical characteristics of themolecule, and the half-life of the molecule.

Preparations for parenteral administration include sterile, aqueous, ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Besides the inert diluents, such compositions can alsoinclude adjuvants, wetting agents, emulsifying, and suspending agents.Further, vasoconstrictor agents can be used to keep the therapeuticagent localized prior to pulsing.

Any cell can be treated by the method of the invention. The illustrativeexamples provided herein demonstrate the use of the method of theinvention for the treatment of tumor cells, e.g., pancreas, lung, headand neck, cutaneous and subcutaneous cancers. Other cell proliferativedisorders are amenable to treatment by the electroporation method of theinvention. The term “cell proliferative disorder” denotes malignant aswell as non-malignant cell populations which often appear to differ fromthe surrounding tissue both morphologically and genotypically. Malignantcells (i.e., tumors or cancer) develop as a result of a multi-stepprocess. The method of the invention is useful in treating malignanciesor other disorders of the various organ systems, particularly, forexample, cells in the pancreas, head and neck (e.g., larynx,nasopharynx, oropharynx, hypopharynx, lip, throat,) and lung, and alsoincluding cells of heart, kidney, muscle, breast, colon, prostate,thymus, testis, and ovary. Further, malignancies of the skin, such asbasal cell carcinoma or melanoma may also be treated by the therapeuticmethod of the invention (see Example 2). Preferably the subject ishuman; however, it should be understood that the invention is alsouseful for veterinary uses in non-human animals or mammals.

In yet another embodiment, the invention provides a method for thetherapeutic application of electroporation to a tissue of a subject fordamaging or killing cells therein while minimizing the amount of painexperienced by the patient. The method includes providing an array ofelectrodes; positioning a second electrode of the array of electrodes inconductive relation to the selected tissue; and applying a firstelectric signal to the first electrode and applying a second electricsignal to the second electrode such that a resultant electrical signalor wave is formed from the wave interference between the first signaland the second. The method may utilize a low voltage and a long pulselength, e.g., a nominal electric field from about 25 V/cm to 75 V/cm andpulse length from about 5 .mu.sec to 99 msec.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

III) EXAMPLES Example 1 Interference Electroporation Treatment In VitroMaterials and Methods

Plasmid.

The plasmid gWiz-Luc, which encoded luciferase, was acquired fromAldevron (Fargo, N. Dak.).

Electroporation.

B16 F10 cells (2.5.times.10.sup.6 cells/mL) were prepared in completemedium (i.e., Mcoy's 5A medium with 10% Fetal Bovine Serum (FBS)). Cellswere split into two groups, Group 1 and Group 2. Group 1 cells weremixed with DNA, but received no interference electroporation (IEP)treatment. Specifically, 1 mL of cell suspension was transferred to acuvette and 50 .mu.L gWiz-Luc (2 mg/mL) was added to this cuvette toarrive at a final concentration of 100 .mu.g/mL of DNA. The cells andDNA were mixed by pipette before placement into a cuvette holder. Thecells received no IEP treatment and the cell suspension was subsequentlytransferred to a 6-well plate. The cells were incubated at 37 degreesCelsius, 5% CO.sub.2 before imaging as described below.

Group 2 cells were mixed with DNA and then received IEP treatment.Specifically, two cuvettes were prepared, in which each cuvette received1 mL of cell suspension and 50 .mu.L gWiz-Luc (2 mg/mL) to arrive at afinal concentration of 100 .mu.g/mL of DNA. A pipette was used to mixthe cell suspension and DNA before placement of each cuvette into acuvette holder. The parameters for IEP were 650V/cm each channel.times.2channels, 100 .mu.s, 1 KHz, 6 pulses. After IEP, 140 .mu.L of cellsuspension was transferred from each cuvette into a respective well of aE-well plate. Each well contained 2 mL complete medium. This was done intriplicate for each cuvette (i.e., 3 wells for each cuvette). The cellswere incubated at 37 degrees Celsius, 5% CO.sub.2 before imaging asdescribed below.

Cell Imaging.

24 hours (hr) after electroporation, medium was removed from each wellof the 6-well plates and 300 .mu.L pre-warmed medium containingD-luciferin (250 .mu.g/ml, Goldbio, St. Louis, Mo., USA) was added toeach well. Cells were then incubated at 37 degrees Celsius for 5 minutes(min) before imaging. Imaging was done with an IVIS Spectrum system(Caliper Life Sciences, Hopkinton, Mass., USA) and assessment ofphotonic emissions from the cells was performed about 8 min to about 10min after incubation of the cells with D-luciferin.

Results

To determine if interference electroporation (IEP) could increase theefficiency of molecule uptake by cells, an in vitro system was utilized,in which cells were split into two different treatment groups. The twotreatment groups were as follows: Group 1 was cells mixed with DNA, butreceiving no IEP treatment; and Group 2 was cells mixed with DNA andreceiving IEP treatment. The DNA used was a plasmid that encodedluciferase and DNA uptake was indirectly measured by luciferaseactivity. Luciferase acts upon its substrate D-luciferin to cause theemission of light or photons.

FIGS. 14 and 15 show the captured images of photonic emission(p/sec/cm.sup.2/sr) from Groups 1 and 2, respectively, 24 hours afterelectroporation. The measurement for each well and the average emissionfor Groups 1 and 2 are shown in Table 1. For each of Groups 1 and 2, theaverage emission was calculated by averaging the measurements from thesix wells depicted in FIGS. 14 and 15, respectively. For FIG. 15, eachrow of three wells corresponded to one of the two cuvettes describedabove that received IEP treatment.

TABLE 1 Measured Photon Emission (p/sec/cm²/sr) Well 1A Well 1B Well 1CWell 2A Well 2B Well 2C Average Group 1 2.93E+05 2.53E+05 2.43E+053.61E+05 3.61E+05 2.32E+05 2.90E+05 Group 2 8.55E+06 6.69E+06 6.74E+061.10E+07 8.58E+06 9.13E+06 8.44E+06

As shown in FIGS. 14 and 15 and Table 1, IEP treatment increasedphotonic emission by 2374% as compared to cells that did not receive IEPtreatment. These data indicated that IEP treatment significantlyincreased DNA uptake by cells. Regular electroporation worked at leastas well as IEP (data not shown).

Example 2 Interference Electroporation Treatment In Vivo Materials andMethods

Plasmid.

The plasmid gWiz-Luc, which encoded luciferase, was obtained fromAldevron (Fargo, N. Dak.). The gWiz-Luc preparation had endotoxin levelsless than 100 EU/mg.

Cells.

B16.F10 melanoma cells were maintained in McCoy's 5A media (Mediatech,Manassas, Va.) supplemented with 10% fetal bovine serum (LifeTechnologies, Grand Island, N.Y.) and 1% Gentamycin at 37 degreesCelsius and 5% CO.sub.2 humidified air.

Mice.

Female C57BL/6J mice (6-8 weeks old) were purchased from JacksonLaboratories (Bar Harbour, Me.).

Tumor Establishment.

50 .mu.L of B16.F10 melanoma cells (1.times.10.sup.6 cells) wereintroduced by subcutaneous (s.c.) injection into the shaved left flankof the C57BL/6J mouse in order to establish the tumor. Each mouse wasmonitored closely for tumor development and tumor volume was measuredusing digital calipers. Tumor volume was calculated by using the formulafor the volume of an ellipsoid: v=.pi.ab.sup.2/6, where a is the longdiameter and b is the short diameter. Tumors were allowed to grow to adiameter of 3 millimeters (mm) to 5 mm (about 5 days after s.c.injection) before delivery of plasmid DNA as described below.

Electroporation.

A tumor was established in each mouse as described above and after thetumor reached a diameter of 3 mm to 5 mm, 50 .mu.g of plasmid DNA (2mg/mL) was injected directly into the tumor using a syringe with a 25gauge needle. The electrode was placed around the tumor and pulses wereapplied using an interference electroporation (IEP) protocol. The IEPprotocol employed a 4-needle electrode with the following parameters:(1) 650 V/channel, 100 .mu.s, 6 pulses, 900 .mu.s gap.

Imaging.

Each mouse was anesthetized and D-Luciferin was injected into the tumor.Imaging was done with an IVIS Spectrum system (Caliper Life Sciences,Hopkinton, Mass., USA). Photonic emissions from the tumor of each mousewas imaged at day 1, day 2, day 5, and day 7 after electroporationtreatment. A portion of the mouse that did not contain the tumor wasalso imaged and served as a control for background photonic emissions.

Results

The data above demonstrated that interference electroporation (IEP)increased the efficiency of DNA uptake by cells in vitro. To furtherexamine the capabilities of IEP, DNA uptake in vivo was examined. Inparticular, tumors were established in two groups of mice and plasmidDNA was administered to each tumor by injection. Group 1 received no IEPtreatment and contained four mice. Group 2 also contained four mice andreceived IEP treatment with the parameters: 650 V/channel, 100 .mu.s, 6pulses, 900 .mu.s gap. Each group of mice was imaged at day 1, day 2,day 5, and day 7 after DNA administration to measure photonic emissionsfrom the tumors.

FIGS. 16, 18, 20, and 22 show the captured images of the photonicemissions (p/sec/cm.sup.2/sr) from the tumors of Group 1 mice at day 1,day 2, day 5, and day 7, respectively. FIGS. 17, 19, 21, and 23 show thecaptured images of the photonic emissions from the tumors of Group 2mice at day 1, day 2, day 5, and day 7, respectively. The solid circleon the hind region of each mouse indicated the area imaged for thetumor. An additional area was imaged on one mouse in each of Group 1 and2 to provide a control for background photonic emission (see solidcircle on the head region of the mouse). Tables 2-5 list themeasurements obtained for each mouse on day 1, day 2, day 5, and day 7,respectively, for Groups 1 and 2. The average photonic emission inTables 2-5 is an average of the photonic emission of the four mice ineach of Groups 1 and 2.

TABLE 2 Measured Photon Emission (p/sec/cm²/sr) on Day 1 Mouse 1 Mouse 2Mouse 3 Mouse 4 Average Group 1 4.88E+05 2.10E+05 2.11E+05 5.41E+053.62E+05 Group 2 8.79E+05 1.49E+06 4.41E+05 2.37E+06 1.29E+06

TABLE 3 Measured Photon Emission (p/sec/cm²/sr) on Day 2 Mouse 1 Mouse 2Mouse 3 Mouse 4 Average Group 1 2.64E+05 1.06E±05 1.18E+05 3.52E+052.10E+05 Group 2 4.51E+05 1.18E+06 9.55E+04 1.31E+06 7.59E+05

TABLE 4 Measured Photon Emission (p/sec/cm²/sr) on Day 5 Mouse 1 Mouse 2Mouse 3 Mouse 4 Average Group 1 1.26E+05 2.52E+04 2.50E+04 1.89E+059.12E+04 Group 2 1.01E+05 2.54E+05 3.76E+04 8.89E+05 3.21E+05

TABLE 5 Measured Photon Emission (p/sec/cm²/sr) on Day 7 Mouse 1 Mouse 2Mouse 3 Mouse 4 Average Group 1 1.33E+05 2.79E+-04 2.12E+04 6.71E+046.23E+04 Group 2 5.11E+04 9.44E+04  4.78E+04 7.39E+05 2.33E+05

FIG. 24 shows a comparison of the average photonic emission for Groups 1and 2 at day 1, day 2, day 3, and day 4. These data indicated that ateach day, IEP treatment increased photonic emission from the tumors ascompared to mice that did not receive IEP treatment. Particularly, IEPtreatment increased photonic emission by 357%, 361%, 351%, and 374% onday 1, day 2, day 5, and day 7, respectively. Accordingly, these datedindicated that IEP treatment significantly increased DNA uptake bytargeted cells (i.e., the tumor cells) in an animal. Regularelectroporation worked at least as well as IEP (data not shown).

Example 3 Interference Electroporation Treatment does not Damage TissueMaterials and Methods

The plasmid gWiz-GFP, which encoded green fluorescent protein (GFP), wasobtained from Aldevron (Fargo, N. Dak.). The gWiz-GFP preparation hadendotoxin levels less than 100 EU/mg.

Cells.

B16.F10 melanoma cells were maintained in McCoy's 5A media (Mediatech,Manassas, Va.) supplemented with 10% fetal bovine serum (LifeTechnologies, Grand Island, N.Y.) and 1% Gentamycin at 37 degreesCelsius and 5% CO.sub.2 humidified air.

Mice.

Female C57BL/6J mice (6-8 weeks old) were purchased from JacksonLaboratories (Bar Harbour, Me.).

Tumor Establishment.

50 .mu.L of B16.F10 melanoma cells (1.times.10.sup.6 cells) wereintroduced by subcutaneous (s.c.) injection into the shaved left flankof the C57BL/6J mouse in order to establish the tumor. Each mouse wasmonitored closely for tumor development and tumor volume was measuredusing digital calipers. Tumor volume was calculated by using the formulafor the volume of an ellipsoid: v=.pi.ab.sup.2/6, where a is the longdiameter and b is the short diameter. Tumors were allowed to grow to adiameter of 3 millimeters (mm) to 5 mm (about 5 days after s.c.injection) before delivery of plasmid DNA as described below.

Electroporation.

A tumor was established in each mouse as described above and after thetumor reached a diameter of 3 mm to 5 mm, 50 .mu.g of plasmid DNA (2mg/mL) was injected directly into the tumor using a syringe with a 25gauge needle. The electrode was placed around the tumor and pulses wereapplied using an interference electroporation (IEP) protocol. The IEPprotocol employed a 4-needle electrode with the following parameters:650 V/channel, 100 .mu.s, 6 pulses, 900 .mu.s gap.

Staining.

Tumor sections were stained with hematoxylin and eosin (H&E) stain.

Results

The above data demonstrated that IEP increased the uptake of DNA bycells both in vitro (i.e., tissue culture) and in vivo (i.e., inestablished tumors in mice). The effect of IEP on cell biology wasfurther examined by H&E staining of tumor sections afterelectroporation. In particular, the staining was utilized to determineif IEP caused tissue damage.

Tumors were established in two groups of mice and plasmid DNA wasadministered to each tumor by injection. Group 1 received no IEPtreatment. Group 2 received IEP after injection. After 7 days, mice weresacrificed and the respective tumors were sectioned for H&E stainingRepresentative staining for Groups 1 and 2 is shown in FIGS. 25 a and 25b, respectively.

As seen in FIGS. 25 a and 25 b, similar cell morphology was observed inthe stained tumor tissue sections from Groups 1 and 2. These dataindicated that tissue receiving IEP treatment was not damaged by theelectrical pulses from the IEP treatment.

Example 4 EPT for Treatment of Tumors In Vivo

A single treatment procedure will involve an injection of bleomycin (0.5units in 0.15 ml saline) intratumorally, using fanning, followed byapplication of six electrical pulses of the first electrical signal andthe second electrical signal, simultaneously, using needle arrayelectrodes as described in the present application, arranged along thecircumference of a circle 1 cm in diameter.

The needle arrays of variable diameters (e.g., 0.5 cm, 0.75 cm and 1.5cm) can also be used to accommodate tumors of various sizes. Stoppers ofvarious heights can be inserted at the center of the array to make thepenetration depth of the needles into the tumor variable. A built-inmechanism will allow switching of electrodes for maximum coverage of thetumor by the pulsed field. The electrical parameters will be: 780 V/cmcenter field strength and 6.times.99 .mu.s pulses spaced at 1-secondintervals.

Example 5 Clinical Trials for Basal Cell Carcinomas and Melanomas

The effectiveness of bleomycin-EPT on tumors will be assessed similar toExample 1.

Example 6 EPT for Head and Neck Cancers

A single-center feasibility clinical study will be conducted in whichthe efficacy of the EPT procedure in combination with intralesionalbleomycin will be compared to that for traditional surgery, radiation,and/or systemic chemotherapy. Approximately 50 study subjects will beenrolled in the study. All study subjects will be assessed prior totreatment by examination and biopsy. Patients will be treated withbleomycin intratumoral injection and needle arrays of differentdiameters with six needles. The voltage will be set to achieve a nominalelectric field strength of 1300 V/cm (the needle array diameter ismultiplied by 1300 to provide the required voltage). The pulse lengthwill be 100 .mu.s. Postoperative assessment of study subjects will beweekly for 4-6 weeks, and monthly thereafter for a total of 12 months.Approximately 8 to 12 weeks following therapy, a biopsy of the tumorsite will be performed. Use of CT or MRI scans will be utilized inaccordance to standard medical follow-up evaluation of HNC subjects.

Tumor evaluation will include measuring the tumor diameter (incentimeters) and estimating its volume (in cubic centimeters). Prior tointratumoral administration of bleomycin sulfate, the tumor site will beanesthetized with 1% lidocaine (xylocalne) and 1:100,000 epinephrine.The concentration of bleomycin sulfate injected will be 4 units permilliliter, up to a maximum dose of 5 units per tumor. If more than onetumor per subject is treated, a total of 20 units per subject should notbe exceeded. The dose of bleomycin administered will be 1 unit/cm.sup.3of calculated tumor volume. Approximately ten minutes subsequent to theinjection of bleomycin sulfate, the applicator will be placed on thetumor and electrical pulses initiated. In this study, success will bedefined as significant tumor regression in a period of 16 weeks or lesswithout major side effects seen with traditional therapy. There are fourpossible response outcomes:

Complete Response (CR): Disappearance of all evidence of tumor asdetermined by physical examination, and/or biopsy.

Partial Response (PR): 50% or greater reduction in tumor volume.

No Response (NR): less than 50% reduction in tumor volume.

If the tumor increases (25% tumor volume) in size, other therapy, ifindicated, will be instituted per subject's desire.

Example 7 Low Voltage Long Pulse Length (LVLP) EPT

Electroporation response of MCF-7 will be carried out at both highvoltage/short pulse length (HVSP) and low voltage/long pulse length(LVLP) using an XTT assay. XTT is a tetrazolium reagent,2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide, which is metabolically reduced inviable cells to a water-soluble formazan product. Therefore, only thecells which are live convert XTT to formazan. The metabolic conversionof XTT to formazan after 70 hours will be measuredspectrophotometrically at 450 nm. (M. W. Roehm, et al., An ImprovedColorimetric Assay for Cell Proliferation and Viability Utilizing theTetrazolium Salt XTT, J. Immunol. Methods 142:2, 257-265, 1991.) Thepercent cell survival values will be calculated using a formula from theO.D. values of the sample. (Control, with 100% cell survival (D-E) andcontrol with 0% cell survival (D-E with SDS).) The experiments with HVSPwill be done to permit direct comparison with the LVLP mode of EPT.

Example 8 Cytotoxicity of Drugs with EPT In Vitro

Cells will be obtained from ATTC (American Type Tissue Collection,Rockville, Md., USA) and maintained by their recommended procedures. Thecells will be suspended in appropriate medium and uniformly seeded in24/96 well plates. One of bleomycin, cisplatin, mitomycin C, doxorubicinand taxol will be added directly to the cell suspensions at finalconcentrations of about 1.times.10.sup.-4 (1E-4) to 1.3.times.10.sup.-9(1.3E-9). The electrical pulses generated by a BTX T820ElectroSquarePorator will be delivered to the cell suspensions inmicroplates using a BTX needle array electrode as described herein.Depending on the experiment, six pulses of either 100 .mu.s or 10 ms andat various nominal electric fields of either high voltage or lowvoltages will be applied between two opposite pairs of a six-needlearray using EPT-196 needle array switch. The microplates will beincubated for either 20 hrs or 70 hrs and the cell survival will bemeasured by the XTT assay.

Example 9 Unipolar Waveform Prototype

Prototypes will be assembled, e.g., using off-the-shelf instruments. Theprototypes will be used to produce two opposing waveforms or signalsthat create an interference waveform. As illustrated in FIGS. 12 a and12 b, in one prototype, each opposing waveform may be unipolar, and, incombination, the resultant interference waveform may also be unipolar asillustrated in FIG. 12 c. In other prototypes, each opposing waveformmay be bipolar as illustrated in FIG. 11, and the resultant interferencewaveform may therefore be bipolar.

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

For example, a variable valve lift (VVL) apparatus or a continuouslyvariable valve lift (CVVL) apparatus implemented so that the valve isoperated with another lift in accordance with revolutions per minute ofan engine, and a variable valve timing (VVT) apparatus which opens andcloses the valve at proper timing in accordance with revolutions perminute of the engine have been researched.

For example, a variable valve lift (VVL) apparatus or a continuouslyvariable valve lift (CVVL) apparatus implemented so that the valve isoperated with another lift in accordance with revolutions per minute ofan engine, and a variable valve timing (VVT) apparatus which opens andcloses the valve at proper timing in accordance with revolutions perminute of the engine have been researched.

What is claimed is:
 1. A method of electroporating cells using thedevice of claim 1, the method comprising: administering selectedmolecules into the cells within the cover area; contacting the cellswith the electrodes; and delivering the one or more electroporatingsignals.
 2. The method of claim 1 further comprising adjusting the coverarea of the electrodes.
 3. The method of claim 1, wherein the one ormore electroporating signals are each associated with an electricalfield, and wherein delivering the one or more electroporating signalsfurther comprises maintaining the electrical field within apredetermined range so as to substantially prevent permanent damage inthe cells within the cover area.
 4. The method of claim 1, wherein theone or more electroporating signals are each associated with anelectrical field, and wherein delivering the one or more electroporatingsignals further comprises maintaining the electrical field within apredetermined range so as to substantially minimize pain.
 5. The methodof claim 1 further comprising adjusting a temperature of the cells toabout 4° C. to about 45° C.
 6. An electroporation device comprising: anapplicator; a plurality of electrodes extending from the applicator; apower supply in electrical communication with the electrodes; and acamera coupled to the applicator and positioned adjacent the electrodes.7. The device of claim 6 further comprising a device memory inelectronic communication with the camera, wherein the device memory isconfigured to store an electroporation treatment database.
 8. Anelectroporation device comprising: an applicator; a plurality ofelectrodes extending from the applicator; a power supply in electricalcommunication with the electrodes; and a cooling/heating element coupledto the applicator and positioned adjacent the electrodes, wherein thepower supply provides a first electrical signal to a first electrode anda second electrical signal to a second electrode, wherein the first andsecond electrical signals combine to produce a wave having a beatfrequency, wherein the first and second electrical signals each have atleast one of a unipolar waveform and a bipolar waveform, wherein thefirst electrical signal has a first frequency and a first amplitude,wherein the second electrical signal has a second frequency and a secondamplitude, wherein the first frequency is different from or the same asthe second frequency, and wherein the first amplitude is different fromor the same as the second amplitude.
 9. The device of claim 8, whereinthe cooling/heating element includes a Peltier cooler.
 10. The device ofclaim 9, wherein the cooling/heating element is configured to adjust atemperature of tumor cells to about 4° C. to about 45° C.